A next generation sequencing technology is able to produce large amounts of data in a short time to remarkably reduce time and cost required for genome decoding as compared to the existing method. In the next generation sequencing technology, sequencing platforms have been developed, and analyzing cost is gradually cheaper over time, and it has succeeded in finding genes responsible for diseases by using the next generation sequencing technology in Mendelian genetic disorder, rare diseases, cancer, etc. According to the most frequently used next generation sequencing of Illumina Inc., DNA is extracted from a sample, and is subjected to mechanical fragmentation, to prepare a library having a specific size, and the library is used for sequencing. Next, initial sequencing data are produced by repeating four kinds of complementary nucleotide binding and isolation reactions with one base unit by using a large sequencing equipment, followed by analysis steps using bioinformatics such as trimming of initial data, mapping, identification of genome mutation, analysis of mutation information (annotation), etc.
The next generation sequencing technology has contributed to creation of new added value through development of innovative drug and industrialization by discovering genome mutations that may have an effect on or may have a possibility of affecting diseases and various biological forms (phenotypes). The next generation sequencing technology is applicable to decoding of DNA as well as RNA and methylation, which makes it possible to perform whole exome sequencing (WES) in which only an exome region encoding protein is captured for sequencing.
Meanwhile, library preparation in the NGS is a process of preparing a library required for sequence analysis by bonding an adapter in the 5′ to 3′ direction in random DNA or cDNA fragment of the sample. The NGS library preparation in the initial stage required complicated processes such as random cleavage of DNA or RNA sample, 3′ and 5′ end repair, adapter ligation, PCR amplification, purification, etc., and a long period of time of 1 day to 2 days. The Illumina, Inc., developed a tagmentation method such as “Nextera XT DNA library Preparation” by improving the initial stage of NGS library preparation. The tagmentation method is a method of treating a composite in which a tag (the existing adapter) is bound to a transposome, in sample DNA, followed by cleavage and adapter ligation at the same time, and amplification by PCR, which reduces time required for preparing the library in 8 samples to 3 hours.
However, when analyzing large amounts of samples, the tagmentation method also requires labor and experimental time of an experimenter since it has a number of steps as follows. The tagmentation method is disadvantageous in that the sequencing is able to be performed after quantifying each DNA of the large amounts of samples to perform tagmentation, confirming a size of a product obtained after the tagmentation, followed by purification, amplification by PCR, purification of an amplified PCR product, and library normalization in the same amount.
Accordingly, the present inventors made an effort to develop a manner in which only desired information is able to be more simply and effectively obtained from a number of samples by reversely considering the concept of NGS obtaining a lot of information from a small number of samples while solving the above-described problems, and as a result, confirmed that when a primer set including all of an adapter sequence, an index sequence, and a target DNA specific-sequence was used, library preparation could be completed by performing PCR once, and completed the present disclosure.